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  Director : Radu CRAINIC (craira@pasteur.fr)


  abstract

 

Our research concerns two positive strand RNA viruses, Enteroviruses and Hepatitis C Virus (HCV). The mechanisms of natural genetic recombination of Enteroviruses was studied on poliovirus model. On the discovery of a correlation between the antigenic and genomic motives we developed a molecular "serotyping" test of Enteroviruses. Studies on HCV were oriented to the understanding of the role of the viral nucleocapsid on the chronic evolution of the infection of humans.



  report

cale

1. Variability and evolution of entEroviruses (F. Delpeyroux, R. Crainic)

Human Enteroviruses (picornaviridae, 64 serotypes) include polioviruses coxsackieviruses, echoviruses, parechoviruses and enteroviruses 68-71. Their transmission is digestive, but the infection of the enteric tract is often sub-clinical. However, they may cause a great variety of severe acute diseases, as meningitis, poliomyelitis, respiratory illnesses, acute myocardiopathy. They sometimes lead to chronic cardiovascular disease, diabetes, acute hemorrhagic conjunctivitis and, perhaps, congenital malformations and fatal neonatal diseases. Poliovirus, the agent of poliomyelitis, is a neurotropic enterovirus of 3 serotypes. (1, 2 and 3). Two vaccines are currently used to protect against poliomyelitis: an injectable - made of inactivated virus (IPV) and an oral - made of attenuated virus (OVP). Only OPV elicits a long-lasting digestive immunity, capable to stop the wild poliovirus transmission.

The genetic variability of RNA viruses (mutation and recombination) is the basis of viral diversity and evolution. By multiplication in the gut of vaccinees, the attenuated, Sabin OPV strains frequently revert to neuropathogenicity In some rare cases, they demonstrate their capacity to induce sporadic vaccine associated paralytic poliomyelitis (VAPP) in vaccinees and/or in their contacts. Moreover, a vaccine-derived poliovirus (VDPV) which reverted to neuropathogenicity and transmissibility, both wild poliovirus-specific characters, induced a poliomyelitis outbreak in Dominican Republic and Haiti and in Philippines. In parallel with our colleagues in CDC (Atlanta), we determined that, before it induced the outbreak, VDPV circulated for about 3 years and had a recombinant genome with another enterovirus (wild poliovirus?) of yet unknown origin.

Natural genetic recombination of poliovirus

In patients with VAPP(S. Guillot, V. Caro, F. Delpeyroux, R. Crainic)

The genetic recombination between the OPV polioviruses is favored by simultaneous multiplication of the three strains of different serotypes (implicitly genotypes) in the gut of the vaccinees. We found that more than 3/4 strains isolated from VAPP cases have an intertypic recombinant genome. In some cases, recombination with wild polioviruses and with other enteroviruses, was also found.

In healthy vaccinees(N. Cuervo, S. Guillot, R. Crainic F. Delpeyroux)

We have investigated the mechanism of genetic recombination among OPV strains (Nancy Cuervo, PhD Thesis. Preferential associations of genomic segments and particular hot-spots of recombinations junctions in the poliovirus genome have been identified. The poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes. The repercussion of the recombination events on the phenotypic changes of OPV strains, and in particular the acquisition of pathogenic characters, is not yet clear.

Molecular taxonomy of enteroviruses(V. Caro, S. Guillot, , R. Crainic, F. Delpeyroux)

To check whether there is a correlation between the antigenicity, the base of the classification of enterovirus serotypes, and their genomic structure, we initiated in 1996 and coordinated a collaborative research project on enterovirus molecular taxonomy. We recently discovered (Valérie Caro, PhD theses) that the gene coding for the protein of capsid VP1, carrying the structure of an important viral neutralization antigenic site, displayed nucleotide sequences in a perfect correlation with enterovirus antigenic properties.

The alignment of nucleotide sequences of a VP1 protein-coding genomic segment allowed the molecular classification of enteroviruses perfectly overlapping the serological classification. This opens the way of molecular identification of enterovirus serotypes. It is probable that the molecular method will replace the presently in use serological method, which is more and more difficult to practice because of the exhaustion of type-specific neutralizing sera.

Phylogeny of epidemic Enteroviruses(A. Szendroi, S. Chevaliez, J. Balanant, G. Berencsi, F. Delpeyroux).

In order to understand the circulation and intra-epidemic evolution of enteroviruses, we analyzed several Echovirus 11 (E11) strains implicated in a severe hemorrhagic syndrome outbreak in Hungary. The Hungarian strains were closely related to each others at the genetic level (99 % nucleotide identity) indicating that they do have a recent common ancestor strain. Comparison with previously or co-circulating E11 isolates from sporadic cases and from environment in Europe have shown that Hungarian strains were closely related to most European strains. A very close relationship was found with Romanian and a Finnish strain isolated from a sporadic hemiparesis case and from environmental waters, respectively. These results prepare the way for the detailed analysis of the viral determinants which differentiate the epidemic E11 from its neighboring strains isolated in non-epidemic period.

2. Physiopathologie of hepatitis C Virus (HCV) infection(directed by Agata Budkowska (P. Maillard, A. Poujol))

The principal characteristics of HCV infection is high incidence of chronic evolution of the disease. HCV core protein has multiple regulatory functions such as modulation of the gene cellular gene expression, regulation of transcription, cellular transformation, apoptosis and lipid metabolism. This protein seems to be responsible for the suppression of the anti-viral T-cell host response during HCV infection.

HCV virion has never been isolated and characterised. We identified in the serum of HCV patients viral particles with properties on non-enveloped HCV nucleocapsids. These particles express on their surface HCV core antigen, contain HCV-RNA and display structure of non-enveloped nucleocapsids by electron microscopy (photo). These viral particles may be detected in serum by immunological methods despite of the presence of circulating anti-HCV antibodies.
We produced similar "nucleocapsid-like" particles in insect cells, infected with recombinant baculovirus bearing genes for the structural HCV proteins. These sub-viral particles, display physicochemical and antigenic properties, and morphology similar to those of non-enveloped nucleocapsids naturally occurring in the serum.

Using monoclonal antibodies, produced by immunisation with serum—derived HCV nucleocapsids we showed for the first time the presence of HCV core antigen in the cytoplasm of hepatocytes of experimentally infected chimpanzees by immunofluorescence and by confocal microscopy. Circulating, non-enveloped HCV nucleocapsids were detected in the serum of both these chimpanzees at the early and viremic phase of the disease (coll. with CDC, Atlanta, USA).

The overproduction of non-enveloped nucleocapsids and their release to the serum from infected cells and the accumulation of the HCV core protein in infected hepatocytes may give to HCV non-conventional means to escape the immune response and to induce immunopathological effects in the infected host. The studies are carried out to determine the structure of HCV nucleocapsid, its immunochemical properties and its interaction with the infected host.

3. RESEARCH AND PUBLIC HEALTH SUPPORT ACTIVITY (S. Guillot, J. Balanant, P. Maillard, A. Budkowska, F. Delpeyroux, R. Crainic)

This activity is formalized by the following structures functioning in our laboratory:

- WHO Collaborative Center for Reference and Research on Poliomyelitis;

- WHO Collaborative Center for Standardization of Viral Vaccines

- the "Study Group for Enteroviruses" (F. Delpeyroux) based on viral laboratories of International Network of Pasteur Institutes involved mainly on research on poliomyelitis and on molecular identification of enteroviruses.

- Regional Reference Laboratory (Europe) and Specialized Laboratory of the WHO Global Polio Laboratory Network for poliomyelitis eradication (together with CDC Atlanta, KTL Helsinki, RIVM Bilthoven, NIBSC London and NIID Tokyo)

- "Study Groups on HCV" (Agata Budkowska) an Inco-Copernicus scientific international collaboration financed by the EU.

Part of the research presented in the previous chapters was the result of international collaborative project within the framework of these structures

 



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
   

BUDKOWSKA Agata , IP, (abudkow@pasteur.fr)

CRAINIC Radu, IP, (craira@pasteur.fr)

DELPEYROUX Francis, INSERM, (delpeyro@pasteur.fr)

CARO Valérie , PhD student (vcaro@pasteur.fr)

CHEVALIEZ Stéphane, Pharmacist, DEA > PhD student (scheval@pasteur.fr )

CUERVO Nancy, PhD student, (ncuervo@pasteur.fr)

POUJOL Aurélie, Stagiaire de recherche

RAKOTO ADRIANARIVELLO Mala, Visitor from IP International Network (Madagascar)

ROMANENKOVA Natalia, Visitor from IP International Network (St. Petersburg)

ONU Adrian, Visitor from IP International Network (Bucharest)

SHERMAN Davin, MD student (USA)

BALANANT Jean, IP, (jbalanan@pasteur.fr)

GUILLOT Sophie, IP, (sguillot@pasteur.fr)

MAILLARD Patrick, IP, (pmaillar@pasteur.fr)

GUIOT Colette, IP


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